Cancer is one of the most life threatening diseases in which cells in a part of the body experience out-of-control growth. According to latest data from American Cancer Society, it is estimated to have 1.6 million new cases of cancer in USA in 2011. Cancer is the second leading cause of death in the United States (second only to heart disease) and will claim more than 570,000 lives in 2011. In fact, it is estimated that 50% of all men and 33% of all women living in the United States will develop some type of cancer in their lifetime. Therefore cancer constitutes a major public health burden and represents a significant cost in the United States. For decades, surgery, chemotherapy, and radiation were the established treatments for various cancers. Patients usually receive a combination of these treatments depending upon the type and extent of their disease. But the chemotherapy is most important option for cancer patient when the surgery treatment is impossible.
Bendamustine, a well known chemotherapy first synthesized in 1963, consists of an alkylating nitrogen mustard moiety and a purine-like benzimidazol moiety with a suggested purine-analog effect (Barman Balfour J A, et al, Drugs 2001; 61: 631-640). Bendamustine has been shown to have substantial activity against low-grade lymphomas (Herold M, et al., Blood, 1999; 94, Suppl 1: 262a), multiple myelomas (Poenisch W, et al., Blood 2000; 96, Suppl 1: 759a), and several solid tumors (Kollmannsberger C, et al., Anticancer Drugs 2000; 11: 535-539). It was also reported that bendamustine effectively induces apoptosis in lymphoma cells (Chow K U, et al., Haematologica, 2001; 86: 485-493). On March 2008, the FDA granted approval to market bendamustine for the treatment of chronic lymphocytic leukemia (CLL). On October 2008, the FDA granted further approval to market bendamustine for the treatment of indolent B-cell non-Hodgkin's lymphoma (NHL) that has progressed during or within six months of treatment with rituximab or a rituximab-containing regimen.
The clinical activity of Bendamustine as a single agent and in combination with other chemotherapeutic and immunotherapeutic drugs, coupled with its potential lack of cross-resistance with many other chemotherapy agents, make bendamustine an attractive therapy for patients with newly diagnosed and refractory hematologic malignancies. [Leoni L M, Semin Hematol. 2011 April; 48 Suppl 1:S4-11]. Currently Bendamustine has about 75 active clinical trials for a variety of cancer indications, such as leukemia, lymphoma, small cell lung cancer, multiple myeloma, MDS, ovarian cancer, breast cancer, and brain tumor. Bendamustine, marketed by Cephalon (TREANDA™), has annual sale of $393 million in US in 2010, and an sale of more than $500 million in US in 2011. The peak sale in 2015 may reach 1 billion $. Bendamustine market exclusive right in US will expire in 2015.
Although Bendamustine has made a significant contribution to cancer treatment, the dose-limiting toxicities and drug resistance remain significant hurdles in its clinical use.
In recent years, histone deacetylases (HDAC) has emerged as an important disease target for cancer treatment [Minucci, S. et al., Nat Rev Cancer 2006, 6, 38-51]. The human HDAC enzymes have 18 isoforms grouped into Class I-IV according to their sequence homology. Class I, II and IV, commonly referred to as the classical HDACs, are comprised of 11 family members. Class III HDACs consists of 7 enzymes and they are distinct from other HDAC family members, therefore are given a unique term sirtuins. The inhibition of HDAC enzyme leads to histone acetylation which is associated with the remodelling of chromatin and plays a key role in the epigenetic regulation of gene expression. In addition, HDAC inhibitors have been shown to evoke the acetylation of many important non-histone proteins such as HSP90, alpha-tubulin, Ku-70, Bcl-6, importin, cortactin, p53, STAT1, E2F1, GATA-1 and NF-kB, which can alter many important signaling networks related to cancer treatment. The underlying mechanism of action of HDAC inhibitors includes the differentiation, cell cycle arrest, inhibition of DNA repair, induction of apoptosis, upregulation of tumor suppressors, down regulation of growth factors, oxidative stress and autophagy. In the last decade, a large number of structurally diverse HDAC inhibitors have been identified and at least 12 HDAC inhibitors are currently in human clinical trials for cancer treatments, including short-chain fatty acid (valproic acid), hydroxamates (SAHA, LBH589, PXD101, JNJ-26481585, ITF2357, CUDC-101), cyclic tetrapeptides (FK-228), benzamide (MS-275), and several other compounds (CHR-3996, 4SC-201, SB939). Among them, SAHA and FK-228 has been approved by the US FDA for the treatment of advanced cutaneous T-cell lymphoma.
In WO/2010/085377, we reported NL-101, a first-in-class dual-functional Bendamustine derivative which potently inhibits the HDAC pathway. The structure of parental drug Bendamustien and NL-101 is shown below:

The biological assay showed that NL-101 potently inhibits HDAC enzyme (HDAC1 IC50 of 9 nM). NL-101 was sent to NCI (NSC#751447) for NCI-60 cell line panel screening. The data showed that NL-101 is about ×25-100 fold more potent than Bendamustine in the NCI-60 cell lines that are representative of a variety of human cancer type. The sixty GI50 values (one for each cell line) make up the fingerprint of the NL-101 and based on this fingerprint, the COMPARE analysis was done by using the COMPARE algorithm on the NCI DTP website. A Pearson correlation coefficient (PCC) of >0.8 indicates >65% agreement in the sensitivity patterns of two compounds and a high likelihood of a common mechanism of action. The COMPARE result showed that the fingerprint of NL-101 did not strongly correlate with any of NSC synthetic compounds (>140,000). In fact, the top match compound is epidoxoform (a doxorubicin derivative) with a PCC of 0.676. Direct comparisons among NL-101, and the conventional nitrogen mustard (e.g. bendamustine, melphalan, and chlorambucil) showed weak correlation coefficients (PCC <0.483). These COMPARE result suggested that NL-101 is not just another conventional nitrogen mustard but possesses unique mechanistic features that differentiate it from the conventional DNA alkylating agents. In another word, NL-101 is expected to be non-cross resistant to the conventional DNA alkylating agents. Therefore NL-101 might have wide potential applications for cancer patients who are resistant, relapse, or refractory to conventional DNA alkylating agents such as bendamustine, melphalan, cisplatin, and temozolomide.
We have developed a first generation formulation of NL-101 for in vivo study, which contains 6 mg/ml NL-101 in buffer system (1.5% acetic acid/0.2% NaOH) with a pH value around 4. The animal study using the first generation formulation of NL-101 shows excellent in vivo efficacy in animal models such as imatinib-resistant Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) model and lung cancer. Ph+ ALL is a leukemia common in adults (˜35% of adult ALL) and carries a poor prognosis. Vincristine (VCR), doxorubicin (Dox), cytarabine (AraC), and cyclophosphamde (CTX) are conventional chemotherapy for Ph+ ALL treatment. Our data showed that single dose of NL-101 (60 mpk) has significantly better in vivo efficacy than the bendamustine, SAHA, VCR, Dox, AraC, and CTX (each dosued at MTD) in imatinib-resistant Ph+ ALL model. Weekly dosing of NL-101 at 60 mg/kg has similar efficacy to Sprycel, which is a FDA approved 2nd line targeted drug for Ph+ ALL treatment. However, the first generation formulation of NL-101 has unfortunately significant disadvantages, such as low pH value, potential precipitation after injection, and series side effects (e.g, damaged mice tail after iv injection and sometime sudden mice death after quick iv injection due to cardiotoxicity). Therefore, there is a strong need to develop a new generation formulation of NL-101 which can overcome the shortcoming of the first generation formulation, particularly the cardiotoxicity, and can be used in future human clinical trials.